Tissue preservation solution, tissue preservation system, and methods of preserving tissue

ABSTRACT

A method for preserving a tissue graft includes submerging the tissue graft in a solution containing one or more antimicrobial and/or stabilizing agents and storing the tissue graft in the solution for a period of time of at least 24 hours while the solution is in an unfrozen state. A tissue preservation system includes a solution containing one or more antimicrobial and/or stabilizing agents and a tissue graft in the solution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of priority to U.S.Provisional Pat. Application No. 63/265,880, filed on Dec. 22, 2021, andU.S. Provisional Pat. Application No. 63/373,827, filed on Aug. 29,2022, the entireties of which are incorporated by reference herein.

TECHNICAL FIELD

The present disclosure relates generally to the fields of neurobiologyand medicine. More particularly, the present disclosure relates tosolutions for preservation of tissue, such as nerve grafts, tissuepreservation systems, and methods of preserving tissue, such as nervegrafts.

BACKGROUND

Nerve damage, regardless of cause, may result in significant, and insome cases severe, discomfort for a subject. Neuropathic injury, inparticular, can cause chronic pain, loss of sensation, loss of some orall muscle control, or other undesirable effects. One potentialtreatment of nerve injuries is surgical intervention via autologoustissue replacement, in which nerve tissue from an uninjured region isgrafted to a damaged region of a nerve. While autologous nerve graftingis considered a gold standard for repair of nerve defects, and inparticular, for repair of peripheral nerve damage, there are significantdisadvantages associated with autologous nerve grafting, such as donorsite trauma, increased complexity of the grafting procedure, scarring,and sensory loss at the donor site, among others.

While neurons of the peripheral nervous system may have an increasedregenerative ability as compared to nerves of the central nervoussystem, many nerve injuries are unable to heal sufficiently withoutsurgical intervention. For example, when an injury results in a lack ofsufficient nerve tissue to allow tension-free healing of the peripheralnerve, surgical intervention may be recommended to increase thelikelihood of successful nerve regeneration and return of sensation ormuscle control. While surgical intervention is quite successful in somecircumstances, the peripheral nerve regeneration process is complex andaffected by multiple factors, such as the microenvironment involved innerve regeneration. Due at least in part to this complexity, there arenumerous circumstances where surgical intervention is less than fullysuccessful.

Allografts offer an alternative to autografts and may be implanted atsites in which there is insufficient nerve tissue present to allowhealing without intervention. Allografts, as well as nerve xenografts,can be processed to provide a suitable substrate for nerve regeneration.However, allografts or xenografts may present challenges. For example,it is frequently desirable to prepare and store allograft or xenografttissue for use as an implant before the implant is needed. However,tissue can experience degradation when stored for relatively longperiods of time. While this degradation can be slowed bycryopreservation, cryopreservation introduces additional and differentcomplications. For example, preservation of tissue at reducedtemperatures requires careful temperature monitoring and introducesincreased costs associated with specialized refrigeration equipmentneeded to transport and store the cryopreserved tissue. Otherpreservation processes, such as tissue lyophilization, involverehydration of tissue and can cause degradation of the tissue whenrehydration fails to adequately restore tissue structures.

SUMMARY

In accordance with the present disclosure, a solution may be useful forpreservation and/or rehydration of tissue, such as a nerve graft. Thesolution may enable room temperature storage of the tissue. The solutionmay be useful for rehydration of lyophilized tissue and may preventdamage to one or more structures of the tissue.

In one aspect, a method for preserving a tissue graft may includesubmerging the tissue graft in a solution containing one or moreantimicrobial and/or stabilizing agents and storing the tissue graft inthe solution for a period of time of at least 24 hours while thesolution is in an unfrozen state.

In another aspect, a method for preserving a tissue graft may includesubmerging the tissue graft in a solution containing chlorhexidinegluconate and propylene glycol and storing the tissue graft in thesolution for a period of time of at least 24 hours, the solution beingin an unfrozen state.

In another aspect, a method for rehydrating a tissue graft may includestoring the tissue graft in a dehydrated state for at least 24 hours,and submerging the tissue graft in a solution containing one or moreantimicrobial and/or stabilizing agents.

In another aspect, a tissue preservation system may include a solutioncontaining one or more antimicrobial and/or stabilizing agents and atissue graft in the solution.

In yet another aspect, a tissue preservation system may include asolution containing chlorhexidine gluconate and propylene glycol and atissue graft in the solution.

Other objects, features, and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the examples,while indicating exemplary embodiments of the present disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to onegeneric formula does not mean that it cannot also belong to anothergeneric formula.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context dictates otherwise. The terms “approximately” and “about”refer to being nearly the same as a referenced number or value. As usedherein, the terms “approximately” and “about” generally should beunderstood to encompass ± 10% of a specified amount or value unlessindicated otherwise in the specification. The use of the term “or” inthe claims and specification is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.” As used herein “another” may mean atleast a second or more.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof the features, as claimed. As used herein, the terms “comprises,”“comprising,” “including,” “having,” or other variations thereof, areintended to cover a non-exclusive inclusion such that a process, method,article, or apparatus that comprises a list of elements does not includeonly those elements, but may include other elements not expressly listedor inherent to such a process, method, article, or apparatus.Additionally, the term “exemplary” is used herein in the sense of“example,” rather than “ideal.” In addition, the term “between” used indescribing ranges of values is intended to include the minimum andmaximum values described herein.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of thedisclosure claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof exemplary embodiments presented herein.

FIG. 1 shows a schematic diagram of an exemplary process for storingtissue or an implant, such as a nerve graft, according to aspects of thepresent disclosure.

FIG. 2 shows a flowchart of an exemplary process for storing tissue oran implant, according to aspects of the present disclosure.

FIG. 3 shows a schematic diagram of another exemplary process forstoring tissue or an implant, according to aspects of the presentdisclosure.

FIG. 4A shows a histological section of nerve tissue stored in asolution, according to aspects of the present disclosure.

FIG. 4B shows a histological section of nerve tissue stored whilefrozen, according to aspects of the present disclosure.

FIG. 4C shows a histological section of nerve tissue stored in asolution, according to aspects of the present disclosure.

FIG. 4D shows a histological section of nerve tissue stored whilefrozen, according to aspects of the present disclosure.

FIG. 5A shows a chart illustrating suture retention characteristics ofnerve tissue stored under different conditions, according to aspects ofthe present disclosure.

FIG. 5B shows a chart illustrating an assessment of endoneurial tubespresent in nerve tissue after being stored in different conditions,according to aspects of the present disclosure.

FIG. 6A shows a histological section of nerve tissue stored in asolution, according to aspects of the present disclosure

FIG. 6B shows a histological section of nerve tissue stored whilefrozen, according to aspects of the present disclosure.

FIG. 6C shows a histological section of nerve tissue stored in asolution, according to aspects of the present disclosure

FIG. 6D shows a histological section of nerve tissue stored whilefrozen, according to aspects of the present disclosure.

FIG. 7A shows a chart illustrating suture retention characteristics ofnerve tissue stored under different conditions, according to aspects ofthe present disclosure.

FIG. 7B shows a chart illustrating an assessment of endoneurial tubespresent in nerve tissue after being stored in different conditions,according to aspects of the present disclosure.

FIG. 8A shows a histological section of nerve tissue that was stored ina solution, according to aspects of the present disclosure.

FIG. 8B shows a histological section of nerve tissue that was storedwhile frozen, according to aspects of the present disclosure.

FIG. 9A shows a histological section of nerve tissue of nerve tissuethat was stored in a solution, according to aspects of the presentdisclosure.

FIG. 9B shows a histological section of nerve tissue of nerve tissuethat was stored while frozen, according to aspects of the presentdisclosure.

FIG. 9C shows a histological section of nerve tissue of nerve tissuethat was stored in a solution, according to aspects of the presentdisclosure.

FIG. 9D shows a histological section of nerve tissue of nerve tissuethat was stored while frozen, according to aspects of the presentdisclosure.

FIG. 9E shows a histological section of nerve tissue of nerve tissuethat was stored in a solution, according to aspects of the presentdisclosure.

FIG. 9F shows a histological section of nerve tissue of nerve tissuethat was stored while frozen, according to aspects of the presentdisclosure.

DETAILED DESCRIPTION

Embodiments of the present disclosure are drawn to tissues, such asnerve grafts, and systems or processes for storing tissue, includingprocesses for storing tissue at room temperature. The nerve grafts maybe stored at room temperature, or in a refrigerated environment. Thenerve grafts may be placed in a solution and stored in a hydrated state.Additionally or alternatively, nerve grafts may be frozen, lyophilized,stored in a lyophilized state, and rehydrated with a solution. The nervegrafts, whether stored in a hydrated state, or after being rehydrated,may be placed on or implanted into a subject, may be useful forlaboratory experimentation, and/or may be useful for demonstrations. Insome aspects, the solution may contain one or more antimicrobial agents.The one or more antimicrobial agents may inhibit grown of one or more ofbacteria, fungus, algae, acanthamoeba, or other impurities. The one ormore antimicrobial agents may include chlorhexidine gluconate. Thesolution may include one or more stabilizing agents. The one or morestabilizing agents may include propylene glycol. The solution mayinclude lactated ringer’s solution (LRS), phosphate buffer saline (PBS),physiological saline, and/or deionized water. The solution mayoptionally be formed from one or more soluble salts. In some aspects,the contents of the solution may be biodegradable.

In some aspects, the tissue, e.g., nerve graft tissue, human orxenogeneic pericardium, intestinal submucosa, dermis, urinary bladdermembranes, epineurium, vascular, or birth tissues, may be processedprior to being introduced to a preserving and/or rehydrating solution.Processed tissue suitable for embodiments of the present disclosure maybe natural or synthetic. For example, the tissue may be soft biologicaltissue obtained from an animal, such as a mammal, including a human or anon-human mammal, or a non-mammal, including a fish, amphibian, orinsect. The graft may be allogeneic or xenogeneic to a subject intowhich the graft is implanted. The tissue may be nerve tissue, including,for example, peripheral nerve tissue or central nervous system tissue.Other types of tissue suitable for the present disclosure include, butare not limited to, epithelial tissue, connective tissue, musculartissue, vascular (e.g., capillary tissue), dermal tissue, skeletaltissue, smooth muscle tissue, cardiac tissue (e.g., pericardium),urological tissue (e.g., urinary bladder membranes), intestinalsubmucosa, birth tissue, ligament tissue, or adipose tissue. Asmentioned above, the soft biological tissue may be mammalian tissue,including human tissue and tissue of other primates, rodent tissue,equine tissue, canine tissue, rabbit tissue, porcine tissue, or ovinetissue. In addition, the tissue may be non-mammalian tissue, selectedfrom piscine, amphibian, or insect tissue. The tissue may be a synthetictissue, such as, but not limited to, laboratory-grown tissue or3D-printed tissue. According to some examples, the tissue is nervetissue obtained from an animal, such as a human or a non-human mammal.The tissue may be obtained and/or treated as disclosed in U.S. Pat.Application No. 17/411,718, entitled “Nerve Grafts and Methods ofPreparation Thereof,” filed on Aug. 25, 2021, the entirety of which isincorporated by reference. The tissue may be an implant and/orbiomaterial as disclosed in U.S. Provisional Pat. Application No.63/292,681, entitled “Nerve Grafts Containing Regenerative Compounds,Methods of Making the Same, and Methods of Treatment Using the Same,”filed on Dec. 22, 2021, U.S. Provisional Pat. Application No.63/265,858, entitled “Nerve Grafts Containing Regenerative Compounds,Methods of Making the Same, and Methods of Treatment Using the Same,”filed on Dec. 22, 2021, U.S. Provisional Pat. Application No.63/265,860, entitled “Drug Delivery System and Methods of Using theSame,” filed on Dec. 22, 2021, and/or U.S. Provisional Pat. ApplicationNo. 63/265,861, entitled “Drug Delivery System and Methods of Using theSame,” filed on Dec. 22, 2021, the entireties of which are incorporatedby reference. In at least some embodiments, an exemplary tissue may be aprocessed human nerve allograph, such as an Avance® Nerve Graft fromAxogen, Inc. (Alachua, FL, US).

Although embodiments of the disclosure are described in relation tonerve grafts for convenience, it is contemplated that other types oftissue, as described above, may be used in the methods and implantsdescribed herein. Further, embodiments of the disclosure focus on theuse of a solution including an antimicrobial agent, such aschlorhexidine gluconate, and a stabilizing agent, such as propyleneglycol, in conjunction with a tissue, but may include differentantimicrobial agents, different stabilizing agents, additionalantimicrobial agents, additional stabilizing agents, or may omit theantimicrobial agent or stabilizing agent.

FIG. 1 illustrates a diagram of an exemplary process 100 for preservingtissue, such as nerve tissue useful as a nerve graft. In one or moreaspects, nerve graft 110 may include one or more neuro-regenerative orimmunosuppressive agents. The one or more neuro-regenerative orimmunosuppressive agents may include an immunophilin ligand. The one ormore neuro-regenerative or immunosuppressive agents may include FK506(tacrolimus), rapamycin (sirolimus), or cyclosporine A. The one or moreneuro-regenerative or immunosuppressive agents may include one or moreother so-called “-olimus” drugs instead of, or in addition to,rapamycin. For example, the one or more neuro-regenerative orimmunosuppressive agents may include temsirolimus, everolimus,ridaforolimus (deforolimus), biolimus, novolimus, zotarolimus, myolimus,and/or amphilimus. The nerve graft may be suitable for implantation in ahuman or non-human animal. FIG. 2 illustrates a flowchart of anexemplary process 200 for producing a preserved tissue, such as nervegraft 110. In particular, process 200 may enable storage of nerve graft110 or other tissue at room temperature, as described below with respectto process 100 (FIG. 1 ).

Process 200 may include obtaining tissue, such as natural or synthetictissue suitable for use with or as a nerve graft, preparing the tissuefor storage, and submerging the tissue in a solution including one ormore antimicrobial agents and/or stabilizing agents. The solution mayenable storage of a nerve graft, e.g., in an unfrozen state, at roomtemperature or refrigerated temperature. As used herein, the phrase“room temperature” refers to temperatures of about 18 degrees Celsius toabout 27 degrees Celsius, while “refrigeration temperature” refers totemperatures of about 0 degrees Celsius to about 18 degrees Celsius. Asused herein, “frozen” refers to a state in which the solution in whichthe tissue is submerged is in a solid state, and “unfrozen” refers to astate in which the solution in which the tissue is submerged is in aliquid state. As used herein, “solution” encompasses both the frozen(solid) state or the liquid state of the mixture.

While process 200 is described in conjunction with process 100 and FIG.1 below, as understood, process 200 may include fewer steps, additionalsteps, and/or different steps as compared to process 100. Additionally,process 200 may include fewer steps, additional steps, and/or differentsteps as compared to each block (e.g., steps 202, 204, and 206)illustrated in FIG. 2 , or the specific order of the steps may bedifferent.

In a step 202 (FIG. 2 ) of process 200, a nerve graft 110 (FIG. 1 ) maybe obtained. A suitable nerve graft may have been harvested from ananimal, such as a mammal, e.g., a human or a non-human mammal, ornon-mammalian tissue as described above. This nerve graft may have beenpreprocessed and may be suitable for immediate use in a subject (e.g.,implantation) or suitable for storage and subsequent use in a subject.In one example, step 202 may include obtaining a nerve graft 110 thathas been processed so as to inhibit an immunogenic reaction in asubject, as well as processed so as to promote proliferation of nervecells from subject tissue into nerve graft 110 following implantation.

In at least some aspects, step 204 may include processing nerve graft110. Suitable processing of this nerve tissue, or other tissue used fornerve graft 110, may include removal of at least some cellular materialor other biological materials. Suitable processing techniques (e.g.,mechanical processing, chemical processing, or a combination of both)may allow a portion or an entirety of the extracellular matrix (“ECM”)to remain intact, such that processed nerve graft 110 forms a scaffoldfor infiltration of nerve cells.

An exemplary processed nerve graft 110 may have been prepared for use asan allograft. This preparation may include harvesting tissue from adonor (e.g., from a cadaver), and processing the tissue, e.g., bydecellularizing the tissue, among other steps. For example, nerve graft110 may be Axogen’s Avance® Nerve Graft. The decellularized tissue mayinclude decellularized material corresponding to an epineurium 112,perineurium 114, and endoneurium 116, such that nerve graft 110 containsone or more structures that correspond to structures of a native nerve.The decellularized tissue may be suitable, in particular, forimplantation in a human for repair of a nerve, e.g., a peripheral nerveinjury. In one or more aspects, the use of a solution including one ormore antimicrobial agents and/or one or more stabilizing agents mayenable storage of nerve graft 110 without significant degradation ofstructures, such as epineurium 112, perineurium 114, and/or endoneurium116.

In order to facilitate use of processed nerve graft 110, decellularizingmay include removal of at least some cellular and noncellular componentsthat may cause or increase an immunological response or other adverseresponse in a subject. Decellularizing may be performed by anycombination of suitable chemical and enzymatic processing. An exemplarymethodology suitable for processing tissue to produce nerve graft 110,including decellularizing to produce acellular nerve tissue, isdescribed in U.S. Pat. No. 9,572,911, entitled “Method forDecellularization of Tissue Grafts,” which issued on Feb. 21, 2017, theentire disclosure of which is incorporated herein by reference. It willbe appreciated, however, that various other methods for preparing tissuespecimens may be used. As understood, steps 202 and 204 may be performedby obtaining preprocessed tissue and/or a preprocessed graft.

Step 206 may include introducing a solution 122 to nerve graft 110 byadding solution 122 to nerve graft 110 or by adding nerve graft 110 tosolution 122. Regardless of whether solution 122 is added to nerve graft110 or nerve graft 110 is added to solution 122, nerve graft 110 may besubmerged in solution 122. As used herein, the term “submerged” meanscompletely or partially surrounded by a fluid. Solution 122 may includeone or more constituents configured to facilitate storage of nerve graft110 by inhibiting degradation of nerve graft 110. Solution 122 mayinhibit degradation of nerve graft 110 by reducing the proliferation ofmicroorganisms. For example, solution 122 may include one or moreantimicrobial agents and/or one or more stabilizing agents. Solution 122may include one or more components, such as a salt solution includingone or more of lactated ringer’s solution (LRS), phosphate buffer saline(PBS), and/or physiological saline, and/or another component, such asdeionized water. The LRS, PBS, physiological saline, and/or deionizedwater may form a majority of solution 122 by weight. Solution 122 mayalso include one or more soluble salts of, for example, magnesium,sodium, calcium, and/or potassium. In particular, solution 122 may beformed from one or more of magnesium sulfate, magnesium chloridehexahydrate, sodium chloride, sodium hydrogen carbonate, calciumchloride, ammonium phosphate dibasic, Trisma hydrochloride, monobasicpotassium phosphate, and/or potassium chloride. When one or more saltsare introduced to solution 122, the salt may have suitable cationic andanionic components, such that the added salt is able to stabilize pH,stabilize protein function, and/or maintain biological activity ofproteins present in nerve graft 110.

When solution 122 includes magnesium sulfate, the magnesium sulfate maybe added to solution 122 in an amount of about 0.01 g/L to about 1 g/L.When solution 122 includes magnesium chloride hexahydrate, the magnesiumchloride hexahydrate may be added to solution 122 in an amount of about0.01 g/L to about 1 g/L. When solution 122 includes sodium chloride, thesodium chloride may be added to solution 122 in an amount of about 5 g/Lto about 50 g/L. When solution 122 includes sodium hydrogen carbonate,the sodium hydrogen carbonate may be added to solution 122 in an amountof about 0.5 g/L to about 5 g/L. When solution 122 includes calciumchloride, the calcium chloride may be added to solution 122 in an amountof about 0.2 g/L to about 0.8 g/L. When solution 122 includes ammoniumphosphate dibasic, the ammonium phosphate dibasic may be added tosolution 122 in an amount of about 0.5 g/L to about 5 g/L. When solution122 includes Trisma hydrochloride, the Trisma hydrochloride may be addedto solution 122 in an amount of about 0.1 g/L to about 1 g/L. Whensolution 122 includes monobasic potassium phosphate, the monobasicpotassium phosphate may be added to solution 122 in an amount of about0.01 g/L to about 0.1 g/L. When solution 122 includes potassiumchloride, the potassium chloride may be added to solution 122 in anamount of about 0.01 g/L to about 0.5 g/L.

Solution 122 may include one or more additives that facilitatepreservation of the structure of nerve graft 110 and/or prevent proteindegradation. For example, solution 122 may include glycerol and/ordimethyl sulfoxide (DMSO). However, in at least some embodiments,solution 122 may be free or substantially free of DMSO.

Solution 122 may be a colorless liquid and may remain colorless in thepresence of nerve graft 110. In some aspects, solution 122 may have alight yellow color. Solution 122 may have a pH between about 4.0 toabout 8.0, or between about 5.0 to about 7.0. Solution 122 may beodorless.

The one or more antimicrobial agents may include chlorhexidinegluconate. Solution 122 may contain chlorhexidine gluconate in an amountof about 0.0005% by weight to about 2% by weight. Solution 122 may bediluted before nerve graft 110 is submerged in solution 122, or whilenerve graft 110 is in the presence of solution 122. Prior to dilution,chlorhexidine gluconate may be present in an amount of about 0.1% byweight to about 10% by weight, in an amount of about 0.2% by weight toabout 5% by weight, or in an amount of about 0.5% by weight to about 2%by weight. Following dilution, chlorhexidine gluconate may be present insolution 122 in an amount of about 0.0005% by weight to about 0.01% byweight, or in an amount of about 0.001% by weight to about 0.005% byweight. The one or more antimicrobial agents may include one or morealcohols or parachlorometaxylenol, either instead of or in addition tochlorhexidine gluconate.

The one or more stabilizing agents may include propylene glycol.Solution 122 may contain propylene glycol in an amount of about 0.001%by weight to about 50% by weight. As noted above, solution 122 may bediluted before nerve graft 110 is submerged in solution 122, or afternerve graft 110 is in the presence of solution 122. Prior to dilution,propylene glycol may be present in an amount of about 1% by weight toabout 30% by weight, or from an amount of about 5% by weight to about15% by weight. Following dilution, propylene glycol may be present in anamount of about 0.001% by weight to about 0.5% by weight, or from about0.01% by weight to about 0.05% by weight. The one or more stabilizingagents may include one or more of propanediol, glycerin, or butyleneglycol, either instead of or in addition to propylene glycol.

When solution 122 includes chlorhexidine gluconate and propylene glycol,solution 122 may include less chlorhexidine gluconate than propyleneglycol, as measured by weight. In particular, the ratio of chlorhexidinegluconate to propylene glycol may be about 1:100 by weight to about 1:2by weight, for example, 1:50 to about 1:4 by weight, or 1:25 to about1:10 by weight. In some aspects, the amount of chlorhexidine gluconatemaybe be about the same as the amount of propylene glycol, (i.e., about1:1 by weight). In some aspects, the ratio of chlorhexidine gluconate topropylene glycol may be about 1:100 by weight, about 1:50 by weight,about 1:25 by weight, about 1:15 by weight, about 1:10 by weight, about1:4 by weight, or about 1:2 by weight. If desired, the amount ofchlorhexidine gluconate may be larger than the amount of propyleneglycol. For example, the ratio of chlorhexidine gluconate to propyleneglycol may be about 2:1 by weight, about 10:1 by weight, about 50:1 byweight, about 100:1 by weight, or about 500:1 by weight.

Nerve graft 110 may be submerged in solution 122 by placing solution 122and nerve graft 110 in a suitable container. This container may include,for example, a vial, a well of a tray, packaging in which nerve graft110 may be stored prior to use, or any other suitable container. In atleast some embodiments, step 206 may include placing the nerve graft110, together with solution 122, in a suitable packaging forwet-preservation of the nerve graft 110. The quantity of solution 122may be based, at least in part, on the size of the container and/or thesize of nerve graft 110. For example, the quantity of solution 122 maybe about 500 µL to about 50 mL, about 500 µL, about 1 mL, about 5 mL,about 10 mL, about 15 mL, about 20 mL, about 30 mL, or about 50 mL. Thepackaging may be translucent or may be opaque, so as to prevent most orall light infiltration.

Following step 206, nerve graft 110 may be prepared for sterilizationand sterilized. As discussed above, nerve graft 110 may be placed withina container together with solution 122, and nerve graft 110 may besterilized while in this container, e.g., a vial, tray, or packaging, inwhich this nerve graft was first submerged in solution 122.Alternatively, nerve graft 110 may be placed within a differentcontainer, e.g., a specialized sterilization container, duringsterilization.

Sterilization may be performed while nerve graft 110 is in the presenceof solution 122. Sterilization may be performed while solution 122 andnerve graft 110 are at room temperature or at a temperature below roomtemperature. In some aspects, nerve graft 110 and solution 122 may betemporarily frozen during the sterilization process. Freezing may beperformed to inhibit microbial action prior to sterilization such that,following sterilization, microbial action is further inhibited by theantimicrobial agent or agents in the sterilized solution 122, or fullyarrested by the antimicrobial agent or agents in the sterilized solution122. Nerve graft 110 and solution 122 may be frozen by reducing thetemperature of nerve graft 110 and solution 122 to below 0° C. followingthe introduction of solution 122 to graft 110, or the introduction ofgraft 110 to solution 122. In particular, nerve graft 110 and solution122 may be held at a temperature of at or above about -20° C., of at orbelow about -20° C., of at or below about -40° C., at or below about-80° C., or at or below about -196° C.

When solution 122 and nerve graft 110 are frozen for sterilization, theconditions under which solution 122 and nerve graft 110 are frozen maybe controlled. For example, the controlled freezing rate may be setbelow about 5° C. per minute, below about 2° C. per minute, for exampleat about 1 degree C per minute or another rate, as desired, in order toprevent rapid temperature reductions and prevent or inhibit theformation of ice crystals.

Sterilization may be performed by one or more appropriate methods. Inone example, nerve graft 110 may be subjected to gamma irradiation 132,as shown in FIG. 1 , in an amount sufficient to sterilize nerve graft110. In some examples, nerve graft 110 may be subjected to a gammairradiation dose of about 0.5 kGy to about 100 kGy, about 1 kGy to about50 kGy, or about 5 kGy to about 30 kGy. The gamma irradiation dose maybe at least about 10 kGy, at least about 15 kGy, at least about 20 kGy,at least about 25 kGy, or at least about 30 kGy. As nerve graft 110 maybe in the presence of solution 122 during sterilization, solution 122may also be sterilized via gamma irradiation 132.

Following sterilization, solution 122 and nerve graft 110 may be storedtogether for a period of time, after which nerve graft 110 may beseparated from solution 122 for use with a subject. In some examples,nerve graft 110 may be stored in solution 122 for a period of at leastone day, at least one week, at least two weeks, at least one month, atleast six weeks, at least two months, at least three months, at leastsix months, or at least one year. In particular, nerve graft 110 may bestored in solution 122 for about 24 hours, about two days, about fourdays, about one week, about two weeks, about four weeks, about sixweeks, about eight weeks, about three months, about six months, aboutone year, about two years, about three years, about five years, orlonger. During this period of time, solution 122 and nerve graft 110 maybe stored at room temperature or at a temperature below roomtemperature. Solution 122 may be stored at a temperature of about 0° C.to about 30° C., of about 12° C. to about 28° C., or about 18° C. toabout 27° C. In some aspects, solution 122 may be stored atrefrigeration temperature, at a temperature of about 4° C. to about 20°C., or at a temperature of about 8° C. to about 10° C. When solution 122and nerve graft 110 are stored at a temperature below room temperature,this temperature may be less than about 18° C. or less than about 12° C.In at least some configurations, solution 122 and the container orpackaging in which solution 122 is stored, may enable visualconfirmation that solution 122 is clear, i.e., free or generally free ofcontamination.

FIG. 3 illustrates an exemplary process 300 for preserving and/orrehydrating tissue, according to one or more aspects of the presentdisclosure. Process 300 may include obtaining a nerve graft 110, asdescribed above with respect to step 202 of process 200 and thecorresponding aspects of process 100. Nerve graft 110 may then besubmerged in a lyophilization solution 142. Lyophilization solution 142may be suitable for lyophilization of nerve graft 110 and may have oneor more constituents that reduce or prevent damage to nerve graft 110during the freeze-drying process.

Once nerve graft 110 is in the presence of lyophilization solution 142,and if desired, following an incubation period, solution 142 and nervegraft 110 may be exposed to freezing temperatures, producing frozennerve graft 120. The temperature of nerve graft 110 and solution 142 maybe reduced at a desired cooling rate to a temperature (e.g., atemperature of about 0° C. to about -80° C.) until nerve graft 110freezes to form frozen nerve graft 120. Following freezing, solution 142may be sublimated, resulting in a dehydrated, or lyophilized, nervegraft 130. Nerve graft 130 may be stored for a desired period of time(e.g., 24 hours, two days, four days, one week, two weeks, four weeks,six weeks, eight weeks, three months, six months, one year, two years,three years, five years, or longer).

Following storage of lyophilized nerve graft 130, nerve graft 130 may berehydrated by being submerged in solution 122. Solution 122, asdescribed with respect to process 300, may include the same constituentsas described above with respect to process 100 and process 200. Inparticular, solution 122 may include one or more antimicrobial agents,such as chlorhexidine gluconate, and one or more stabilizing agents,such as propylene glycol. As described above, solution 122 may includeLRS, PBS, physiological saline, and/or deionized water.

In some aspects, nerve graft 130 may be rehydrated using a rehydrationsolution that is different than solution 122. This rehydration solutionmay include one or more of deionized water, saline, LRS, or PBS. Nervegraft 130 may be immersed in this rehydration solution for a suitableperiod of time. Once nerve graft 130 is adequately rehydrated, therehydration solution may be removed and replaced with solution 122 forpreserving the rehydrated nerve graft 140 and preserving nerve graft140. Such a rehydrated nerve graft 140 may be suitable for demonstrationpurposes, scientific experimentation, or implantation into a human ornon-human subject.

Nerve graft 110, described above with respect to process 100 and process200, and nerve graft 140, described above with respect to process 300,may be used in methods for treating a human or non-human animal subject.These methods may include implantation of nerve graft 110 or 140 to aninjury site. A method of using nerve graft 110 or 140, whether stored atroom temperature or in a lyophilized form, may include preparing adamaged nerve for implantation of nerve graft 110 or 140. Preparationmay include exposing an injured nerve, preparing a nerve bed, anddebriding and cleaning the damaged nerve to form a proximal nerve endand a distal nerve end. A nerve graft 110 or 140 having a desireddiameter and/or length may be selected from a plurality of nerve grafts110 or 140 having different diameters and/or lengths. A nerve graft 110or 140 having a suitable length may be determined based on the distancebetween proximal and distal nerve ends, and may be approximately equalto this distance. Similarly, the diameter of nerve graft 110 or 140 maybe selected based on the diameters of the proximal and distal nerveends. The selected nerve graft 110 or 140 may then be removed from astorage system or container (e.g., packaging). This storage system orcontainer may correspond to the above-described storage systems forstorage of nerve graft 110 and may include solution 122. If desired, thenerve graft 110 or 140 may be rinsed in sterile saline or water forimplantation after being removed from the storage system or container.If the case of nerve graft 140, which has been lyophilized, nerve graft140 may be rehydrated and submerged in solution 122, either as part ofrehydration or subsequent to rehydration. If nerve graft 110 or 140 islonger than the distance between the proximal and distal nerve ends,nerve graft 140 may be trimmed to a length that is approximately equalto this distance.

Implantation of nerve graft 110 or 140 may be performed by suturingnerve graft 110 or 140 to a proximal nerve end and a distal nerve end tobridge the gap between these nerve ends, resulting in an implanted nervegraft 140. The surgical site with the implanted nerve graft 110 or 140may be sutured closed. The implanted nerve graft 110 or 140 mayfacilitate recovery of the subject’s tissue. In particular, nerve graft110 or 140, after implantation, may provide structural support thatenables axonal regeneration and healing of the injured nerve tissue.

EXAMPLES

The disclosure may be further understood by the following non-limitingexamples. The examples are intended to illustrate embodiments of theabove disclosure, and should not be construed as to narrow its scope.One skilled in the art will readily recognize that the examples suggestmany other ways in which the embodiments of the disclosure could bepracticed. It should be understood that numerous variations andmodifications may be made while remaining within the scope of thedisclosure.

Example 1

Nerve grafts were prepared by decellularizing nerve tissue and dividingthe nerve tissue into two storage conditions. The decellularized nervegrafts were prepared for storage at room temperature. Nerve grafts werealso prepared for storage while remaining in a frozen state forcomparison with the nerve grafts stored at room temperature. Afterstorage, these nerve grafts were observed to exhibit microstructure andobserved to exhibit suture pull out strength similar to currentlymarketed nerve grafts. Accordingly, the microstructure and suture pullout strength were consistent with suitability for supporting peripheralnerve regeneration in a human or non-human subj ect.

The nerve grafts were further divided into two groups. The first groupof nerve grafts included four nerve grafts, two nerve grafts having adiameter of greater than 3 mm, and two nerve grafts having a diameterless than 3 mm, which were submerged in a preservative solution andstored at room temperature. A second group of nerve grafts included fournerve grafts, two nerve grafts having a diameter of greater than 3 mm,and two nerve grafts having a diameter less than 3 mm, which were placedin a small amount of LRS and stored in a frozen condition.

The nerve grafts of the first group, for storage at room temperature,were submerged in a diluted preservative solution. This dilutedpreservation solution was formed by mixing 5.2 mL of a concentratedsolution containing chlorhexidine gluconate and propylene glycol with1000 mL of LRS as a diluent. The diluted solution included 0.005%chlorhexidine gluconate by weight, and 0.05% propylene glycol by weight.Each nerve graft of the first group together with the diluted solution,was frozen at -80° C. The frozen nerve grafts and solution were thensterilized via gamma irradiation. The gamma-irradiated nerve grafts werethawed and allowed to warm to room temperature. These nerve grafts wereincubated, at room temperature, for a period of two weeks while beingprotected from light. The nerve grafts belonging to the second groupremained frozen after gamma irradiation. The frozen nerve grafts werestored in the frozen condition for the same two-week period of time,while being protected from light. Once the two-week incubation wascompleted, the frozen nerve grafts were thawed, allowed to warm to roomtemperature, and evaluated as described below.

The evaluation of each nerve graft included comparing each individualnerve graft to a plurality of acceptance criteria to determine whetherthe nerve graft would be suitable for use as an implant followingpreservation and storage. In particular, each individual nerve graftfrom the two groups was visually inspected, including the nerve graftitself and the storage solution. Each sample was handled to determinestiffness. Each nerve graft was evaluated via anti-lamininimmunohistochemistry to observe and evaluate endoneurial tube structure(results shown in FIGS. 4A-4D and 5B), and tested to determine suturepull-out strength (results shown in FIG. 5A). The frozen nerve graftsfrom the second group were thawed prior to these evaluations.

During the visual inspection of the solution for the roomtemperature-stored nerve grafts, it was observed that each solution wasclear and free of visible contamination. The room temperature-storednerve grafts were not visually inferior to the frozen nerve grafts.During physical handling, the room temperature-stored nerve grafts weresomewhat stiffer than the nerve grafts stored in a frozen condition, butretained suitable flexibility.

Results of the histological evaluations are summarized in FIGS. 4A-4D.Each of FIGS. 4A-4D illustrates a laminin-stained section of a nervegraft, in which endoneurial tube basement membranes containing Lamininprotein are identified by dark brown stain deposition (darker areas inFIGS. 4A-4D) inside of the fascicles of the nerve tissue. FIG. 4Aillustrates a nerve graft stored at room temperature, the nerve grafthaving a diameter greater than 3 mm. In FIG. 4A, each of thesubstantially circular portions illustrates a preserved endoneurial tubestructure inside of the fascicle. FIG. 4B illustrates a nerve grafthaving a diameter greater than 3 mm that was stored in a frozencondition. FIGS. 4C and 4D illustrate nerve grafts stored at roomtemperature and nerve grafts stored in a frozen condition, respectively.The nerve grafts illustrated in FIGS. 4C and 4D each had a diameter ofless than 3 mm. As can be seen in FIGS. 4A and 4C, some tissueseparation was observed in the fascicles. Some nerve graft samplesstored at room temperature exhibited minor changes in endoneurial tubesize and shape. However, the room temperature-stored nerve graftscontained substantially intact endoneurial tubes in the fascicles. Theendoneurial tubes were observed to have experienced relatively limitedstructural changes and had a morphology consistent with sufficientpreservation. The preservation of the endoneurial tubes in theroom-temperature stored nerve grafts was comparable to that of the nervegrafts stored while frozen.

FIGS. 5A and 5B illustrate exemplary quantitative analyses of the nervetissues that were stored as described above. FIG. 5A shows the resultsof suture pull-out tests as a bar chart in which each bar representingthe range of measured values for the maximum force that was needed toseparate a suture, with the smallest maximum pull-out force representedby the bottom of each bar and a largest maximum pull-out forcerepresented by the top of each bar. The error bars in FIG. 5A illustratethe standard deviation, the “X” illustrates the mean value, and thehorizontal line within each bar illustrates the median value of thesetests.

The suture pull-out assessment, the results of which are shown in FIG.5A, was performed with a 9-0 suture, suitable for implantation of thistype of nerve graft, that was inserted through the epineurium about 5 mmfrom the edge of each nerve graft, and a loop was formed for attachmentof the suture to a mechanical testing device. A constant pull force of 1mm per second was applied until suture or tissue failure. The maximumforce applied indicates the relative strength of the preserved tissuebeyond the strength of the sutures that may be used for implantation. Ascan be seen in FIG. 5A, the room temperature-stored nerve grafts hadsuitable maximum pullout forces, forces in excess of the strength ofsuture material, of greater than about 0.5 N (Newtons) and less thanabout 1.2 N, as compared to nerve grafts stored while frozen, whichexhibited maximum pullout forces of about 0.95 N. While the roomtemperature-stored tissue had a somewhat higher variability in thevalues measured, the measured values were suitable for implantabletissue. While not wishing to be bound by theory, this variability may bedue to the relatively higher stiffness of the samples having a diameterless than 3 mm and/or suturing variability. In all measured samples,suture failure was observed before the tissue failure, which indicatesacceptable tissue preservation.

FIG. 5B illustrates the result of endoneurial tube assessments that wereperformed on the nerve graft of each group. The endoneurial tubeassessment (ETA) reflected in FIG. 5B includes a quantitative measure ofthe endoneurial tube perimeter per 100,000 µm² of the fascicular area.The laminin-stained surfaces identify endoneurial tube basementmembranes, higher scores being assigned to nerve grafts containinglarger perimeters of the endoneurial tubes that fit into a defined rangeof size and circularity. The bars in FIG. 5B include a first bar havinga height that represents the average ETA score for the nerve graftsstored in a frozen state and a second bar that has a height thatrepresents the average ETA score for the nerve grafts that were storedat room temperature. Nerve grafts that were stored in a frozen conditionhad somewhat higher ETA scores as compared to the roomtemperature-stored nerve graft samples. As shown in FIG. 5B, the averageETA score for nerve grafts stored in a frozen condition was about12,000, while the ETA score for the room temperature-stored nerve graftswas about 8,200. The ETA scores for the room temperature-stored nervegrafts, while smaller than the nerve tissue stored while frozen,remained within an acceptable range. In some aspects, these ETA scoreswere consistent with nerve grafts that, after storage at roomtemperature, are able to satisfy acceptance criteria for implantation ina human or non-human subject and thus indicated appropriate preservationof the room temperature-stored nerve grafts.

Example 2

Nerve grafts were prepared by decellularizing nerve tissue. Eachdecellularized nerve was split between four groups, as described below.The decellularized nerve grafts were prepared for storage at roomtemperature. Decellularized nerve grafts were also prepared for storagein a frozen state, these nerve grafts being suitable for implantation ina human or non-human subject. A first group of nerve grafts in Example 2included 12 nerve grafts, 6 nerve grafts having a diameter of greaterthan 3 mm, and 6 nerve grafts having a diameter less than 3 mm. Allnerve grafts of the first group were submerged in a preservativesolution and stored at room temperature for 4 weeks. A second group ofnerve grafts included 12 nerve grafts, 6 nerve grafts having a diameterof greater than 3 mm, and 6 nerve grafts having a diameter less than 3mm, that were placed in LRS and stored in a frozen condition for 4weeks. The third group included 12 nerve grafts, 6 nerve grafts having adiameter of greater than 3 mm, and 6 nerve grafts having a diameter lessthan 3 mm, that were submerged in a preservative solution and stored atroom temperature for 12 weeks. A fourth group of nerve grafts included12 nerve grafts, 6 nerve grafts having a diameter of greater than 3 mm,and 6 nerve grafts having a diameter less than 3 mm, that were placed inLRS and stored in a frozen condition for 12 weeks.

The nerve grafts of the first and third groups, which were stored atroom temperature, were introduced to a diluted preservative solutionprior to storage. This diluted solution was formed by mixing 3.13 mL ofa concentrated solution containing chlorhexidine gluconate and propyleneglycol with 1000 mL of LRS. The diluted solution included 0.003%chlorhexidine gluconate by weight, and 0.03% propylene glycol by weight.Each nerve graft of the first group, including the diluted solution, wasthen frozen and sterilized via gamma irradiation, as described abovewith respect to Example 1. The gamma-irradiated nerve grafts were thenthawed and allowed to warm to room temperature. The room-temperaturenerve grafts of the first and third groups were incubated, at roomtemperature, for a period of 4 weeks or 12 weeks, respectively, whilebeing protected from light. The nerve grafts belonging to the secondgroup and the fourth group were stored while frozen and protected fromlight for 4 weeks or 12 weeks, respectively.

Each nerve graft was evaluated and compared to acceptance criteriadescribed above with respect to Example 1. During the visual inspectionof the solution of the room temperature-stored nerve grafts, it wasobserved that each solution was clear and free of visible contamination.The room temperature-stored nerve grafts were not visually inferior tothe frozen nerve grafts, and, while observed during handling to bestiffer than the nerve grafts stored in a frozen condition, exhibitedsuitable flexibility.

Exemplary results of the histological evaluations for each of the fourgroups are shown in FIGS. 6A-6D. Each of FIGS. 6A-6D illustrates alaminin-stained section of a nerve graft, having an enlarged (FIGS.6A-6D having increased magnification to facilitate observation of nervegraft structure) view as compared to FIGS. 4A-4D. FIG. 6A illustrates anerve graft of the first group, stored at room temperature for 4 weeks.FIG. 6B illustrates a nerve graft of the second group that was stored ina frozen condition for four weeks. FIGS. 6C and 6D illustrate nervegrafts stored at room temperature (third group) and nerve grafts storedin a frozen condition (fourth group), respectively. The nerve graftsillustrated in FIGS. 6C and 6D were each stored for 12 weeks. As can beseen in FIGS. 6A and 6C, the room temperature-stored nerve graftscontained intact endoneurial tubes that were comparable in structure tothe nerve grafts that were stored in a frozen state. Each individualendoneurial tube experienced relatively minimal structural changes andsufficient preservation, as compared to the nerve grafts of FIGS. 6B and6D, which were stored while frozen.

FIGS. 7A and 7B illustrate exemplary quantitative analyses of the nervetissues that were stored as described above. FIG. 7A shows the resultsof suture pull-out tests as a bar chart in which the height of each barillustrates the mean pull out force, in Newtons, that was measured foreach group. The error bars in FIG. 7A illustrate the standard deviationof these measurements. In FIG. 7A, “Frozen T1” refers to the group ofnerve grafts that were frozen and stored for 4 weeks, “Room Temp T1”refers to the group of nerve grafts that were stored at room temperaturefor 4 weeks, “Frozen T2” refers to the group of nerve grafts that werefrozen and stored for 12 weeks, “Room Temp T2” refers to the group ofnerve grafts that were stored at room temperature for 12 weeks.

As can be seen in FIG. 7A, the room temperature-stored nerve graftsperformed similarly to the corresponding groups of nerve grafts thatwere stored while frozen. The frozen and room temperature-stored nervegrafts stored for 4 weeks exhibited an average suture pull-out force ofbetween about 0.7 N and about 0.8 N. The nerve grafts stored in a frozencondition for 12 weeks exhibited an average suture pull-out force ofbetween about 0.8 N and about 0.9 N, while the nerve grafts stored in atroom temperature showed an average suture pull-out force of betweenabout 0.9 N and about 1.0 N. All tests resulted in suture failure priorto failure of the nerve graft, including the tests for each nerve graftstored at room temperature, indicating satisfactory preservation.Additionally, variability across these measurements was acceptable forthe group of nerve grafts stored for 4 weeks, and the group of nervegrafts that were stored for 12 weeks.

FIG. 7B illustrates the result of endoneurial tube assessments (ETAs)that were performed on each nerve graft to evaluate endoneurial tubeperimeter, as described above, with higher scores indicating largerperimeter of endoneurial tubes. The bars in FIG. 7B include a first bar(“Frozen T1”) having a height that represents the average ETA score forthe nerve grafts stored in a frozen state for 4 weeks. This ETA scorewas about 10,000. A second bar (“Room Temp T1”) in FIG. 7B has a heightthat represents the average ETA score for nerve grafts that were storedat room temperature for 4 weeks, about 9,000. FIG. 7B further includes athird bar (“Frozen T2”) having a height that represents the average ETAscore for the nerve grafts stored in a frozen state for 12 weeks, about13,000, and a fourth bar (“Room Temp T2”) having a height thatrepresents the average ETA score for nerve grafts that were stored atroom temperature for 12 weeks, about 7,500. Nerve grafts that werestored at room temperature exhibited comparable ETA scores to nervegraft samples stored in a frozen condition for the same period of time.The ETA scores for the room temperature-stored nerve grafts remainedwithin a suitable range for the group of nerve grafts stored for 4weeks, and for the group of nerve grafts stored for 12 weeks.Specifically, ETA scores for the room temperature-stored nerve grafts,whether stored for 4 weeks or 12 weeks, remained within an acceptablerange. In some aspects, these ETA scores were consistent with nervegrafts that, after storage at room temperature, are able to satisfyacceptance criteria for implantation in a human or non-human subject andthus indicated appropriate preservation of the room temperature-storednerve grafts.

Example 3A

Nerve grafts were prepared by decellularizing nerve tissue and dividingthe nerve tissue into two groups with different storage conditions. Thedecellularized nerve grafts for the first group were prepared forstorage at a refrigeration temperature of 4° C. Nerve grafts for thesecond group were prepared for storage while remaining in a frozen statefor comparison with the nerve grafts stored at the refrigerationtemperature. The first group of nerve grafts included ten nerve grafts,five nerve grafts having a diameter of greater than 3 mm, and five nervegrafts having a diameter less than 3 mm, which were each submerged in apreservative solution and stored at the refrigeration temperature of 4°C. The second group of nerve grafts included ten nerve grafts, fivenerve grafts having a diameter of greater than 3 mm, and five nervegrafts having a diameter less than 3 mm, each of which were placed in asmall amount of LRS and stored in a frozen condition at -80° C.

The decellularized nerve grafts were prepared for storage at 4° C. bybeing placed in a diluted preservative solution. This diluted solutionwas formed by mixing 5.2 mL of a concentrated solution containingchlorhexidine gluconate and propylene glycol with 1000 mL of phosphatebuffer saline (PBS) as a diluent. The PBS had a pH of 7.4. The dilutedsolution included 0.005% chlorhexidine gluconate by weight, and 0.05%propylene glycol by weight. The solution also contained dissolvedmagnesium chloride hexahydrate in an amount of 0.6% by weight.

Each nerve graft was then frozen and sterilized via gamma irradiation.The gamma-irradiated nerve grafts of the first group were then thawed ina refrigerated environment and allowed to warm to the refrigerationtemperature of 4° C. The refrigeration-temperature nerve grafts wereincubated, at 4° C., for a period of 12 weeks while being protected fromlight.

Each nerve graft was evaluated and compared to acceptance criteriadescribed above with respect to Example 1. During the visual inspectionof the solution of the refrigeration-temperature-stored nerve grafts, itwas observed that each solution was clear and free of visiblecontamination. The refrigeration-temperature-stored nerve grafts werenot visually inferior to the frozen nerve grafts, and contained onlysmall amounts of sediment in some of the storage containers.Additionally, the refrigeration-temperature-stored nerve graftsexhibited similar mechanical integrity when compared to the frozen nervegrafts or to the samples from Example 1 and Example 2.

Histology was also performed on the nerve grafts after the 12-weekincubation period to assess endoneurial tube structure. Exemplaryresults of the histological evaluations for the refrigerated and frozensamples are shown in FIGS. 8A and 8B, respectively. FIG. 8A illustratesa laminin-stained section of a nerve graft that was stored in arefrigerated condition. FIG. 8B illustrates a laminin-stained section ofa nerve graft that was stored in a frozen condition. FIGS. 8A and 8Beach illustrate preserved endoneurial tube structure inside of a singlefascicle. As can be seen in FIGS. 8A and 8B, laminin staining wassomewhat reduced as compared to Examples 1 and 2, resulting in asomewhat lighter visual appearance for both the refrigerated and frozensamples. Both the refrigerated and frozen samples had acceptableappearances and substantially intact endoneurial tubes in the fascicles.

A quantitative analysis of the nerve tissues of Example 3 is provided asTable 1 below. Table 1 presents ETA scores for four exemplary samples ofeach group. Results for the first group are presented in the columnlabelled “Refrigerated,” while results for the second group arepresented in the column labelled “Frozen.” Table 1 presents one ETAscore for a sample having a diameter greater than 3 mm that was storedin a refrigerated condition, and one ETA score for a sample having adiameter greater than 3 mm that was stored in a frozen condition. Threeadditional ETA scores are included for exemplary samples havingdiameters of less than 3 mm that were stored in a refrigeratedcondition, as well as three ETA scores for samples having diameters lessthan 3 mm that were stored in a frozen condition.

As indicated in Table 1, the ETA scores for all refrigerated sampleswere acceptable, regardless of whether the diameter of the sample wasgreater than 3 mm or less than 3 mm. The scores for the first group werecomparable to the scores for the samples stored in a frozen (-80 degreeCelsius) condition. The average ETA score for the refrigerated sampleswas slightly smaller than the average ETA score for the frozen samples.

TABLE 1 Frozen Refrigerated Sample 1 (size > 3 mm) 6,351.3.00 8,299.2.00Sample 2 (size < 3 mm) 3,988.1.00 4,500.7.00 Sample 3 (size < 3 mm)7,561.2.00 8,802.1.00 Sample 4 (size < 3 mm) 11,385.7.00 7,020.2.00Average 7,321.6.00 7,155.6.00

The properties of the refrigeration-temperature-stored nerve grafts ofExample 3A, represented at least in part by the above-identifiedevaluations, were consistent with nerve grafts that, after storage atroom temperature, are consistent with suitability for supportingperipheral nerve regeneration in a human or non-human subject afterpreservation for 12 weeks, and thus indicated appropriate preservationof the refrigeration-temperature-stored nerve grafts.

Example 3B

Nerve grafts were prepared and stored as described above with respect toExample 3A. A first group of nerve grafts included eight nerve graftsprepared in this manner, the first group including four nerve graftshaving a diameter of greater than 3 mm, and four nerve grafts having adiameter less than 3 mm, each of which was submerged in a preservativesolution (described above with respect to Example 3A) and stored at therefrigeration temperature of 4° C. A second group of nerve graftsincluded eight nerve grafts, four nerve grafts having a diameter ofgreater than 3 mm, and four nerve grafts having a diameter less than 3mm, that were each placed in a small amount of LRS and stored in afrozen condition at -80° C. All nerve grafts were sterilized andincubated at either refrigeration temperatures of 4° C., or at -80° C.,for a period of two weeks.

Following incubation, histology was performed to assess endoneurial tubestructure. Exemplary results of the histological evaluations for therefrigerated and frozen samples are shown in FIGS. 9A-9F. FIG. 9Aillustrates a laminin-stained section of a nerve graft that was storedin a refrigerated condition, the nerve graft having a diameter that isgreater than 3 mm. FIG. 9B illustrates a laminin-stained section of anerve graft that was stored in a frozen condition, the nerve grafthaving a diameter greater than 3 mm. FIG. 9C is an enlarged image of anerve graft stored at refrigerated temperature, and FIG. 9D is anenlarged image of a nerve graft that was stored while frozen.

As can be seen in FIGS. 9A-9D, the nerve graft samples stored atrefrigeration temperatures (FIGS. 9A and 9C) presented substantiallyintact endoneurial tube structure. The nerve graft samples stored atrefrigeration temperatures exhibited slightly looser perineurial ECM,slightly increased fascicular separation, and some non-specific stainingin the endoneurial tubes as compared to samples that were stored in thefrozen condition. Despite this, endoneurial tubes of the refrigeratedsamples were observed to have experienced relatively limited structuralchanges and a morphology (e.g., microstructure) consistent withsufficient preservation. Overall, the preservation of the endoneurialtubes in the refrigeration-temperature stored nerve grafts was visuallycomparable to that of the nerve grafts stored while frozen.

Endoneurial tube assessments (ETA) were also performed on therefrigerated and frozen samples having diameters greater than 3 mm. Thesamples stored in a refrigerated condition exhibited an average ETAscore of 9,359.60.00 (with a standard deviation of 2,306.95.00), ascompared to an average ETA score of 14,493.04.00 (with a standarddeviation of 1,670.25.00). The ETA scores of the refrigerated sampleswere consistent with nerve tissue that, after storage, are able tosatisfy acceptance criteria for implantation in a human or non-humansubject.

FIGS. 9E and 9F illustrate laminin-stained sections of nerve grafts thatwere stored at refrigeration temperatures (FIG. 9E) and at -80° C. (FIG.9F). The nerve grafts illustrated in FIGS. 9E and 9F each had a diameterof less than 3 mm. As can be seen by comparing FIGS. 9E and 9F, therefrigeration-temperature-stored nerve grafts experienced minorseparation of fascicular tissue from the surrounding tissue.Additionally, as shown in FIG. 9E, some endoneurial tube damageoccurred, and some shrinkage was observed. These alterations inmorphology were limited to samples having a diameter of less than 3 mmand were relatively minor in nature. The nerve grafts stored at 4° C.were observed to exhibit microstructure consistent with suitability forsupporting peripheral nerve regeneration in a human or non-human subjectafter preservation, and thus indicated appropriate preservation of therefrigeration-temperature-stored nerve grafts.

It should be understood that although the present disclosure has beenmade with reference to preferred embodiments, exemplary embodiments, andoptional features, modifications and variations of the concepts hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis disclosure as defined by the appended claims. The specificembodiments and examples provided herein are examples of usefulembodiments of the present disclosure and are non-limiting andillustrative only. It will be apparent to one skilled in the art thatthe present disclosure may be carried out using a large number ofvariations of the devices, device components, methods, and steps setforth in the present description. As will be recognized by one of skillin the art, methods and devices useful for the present methods caninclude a large number of various optional compositions and processingelements and steps.

What is claimed is:
 1. A method for preserving a tissue graft, themethod comprising: submerging the tissue graft in a solution containingone or more antimicrobial and/or stabilizing agents; and storing thetissue graft in the solution for a period of time of at least 24 hourswhile the solution is in an unfrozen state.
 2. The method of claim 1,wherein the tissue graft is a nerve graft.
 3. The method of claim 1,wherein the solution contains one or more antimicrobial agents, the oneor more antimicrobial agents comprising chlorhexidine gluconate.
 4. Themethod of claim 3, wherein the chlorhexidine gluconate is present in thesolution in an amount of about 0.0005% by weight to about 2% by weight.5. The method of claim 1, wherein the solution contains one or morestabilizing agents, the one or more stabilizing agents comprisingpropylene glycol.
 6. The method of claim 5, wherein the propylene glycolis present in the solution in an amount of about 0.001% by weight toabout 50% by weight.
 7. The method of claim 1, wherein the solutionfurther comprises one or more of lactated ringer’s solution (LRS),phosphate buffer saline (PBS), physiological saline, or deionized water.8. The method of claim 1, wherein the solution is free of dimethylsulfoxide.
 9. The method of claim 1, wherein the solution issubstantially free of dimethyl sulfoxide.
 10. The method of claim 1,wherein the solution further comprises one or more of glycerol ordimethyl sulfoxide.
 11. The method of claim 1, wherein the tissue graftis stored at a temperature of about 0° C. to about 30° C.
 12. The methodof claim 1, wherein the tissue graft is stored at room temperature. 13.The method of claim 1, further comprising sterilizing the tissue graftprior to storing the tissue graft.
 14. The method of claim 13, whereinsterilizing the tissue graft comprises exposing the tissue graft togamma irradiation.
 15. The method of claim 13, further comprisingfreezing the tissue graft and the solution prior to sterilizing thetissue graft.
 16. The method of claim 15, further comprising thawing thesolution to the unfrozen state after sterilization and before storingthe tissue graft.
 17. The method of claim 1, wherein the solution isformed from one or more soluble salts of magnesium, calcium, sodium, orpotassium.
 18. The method of claim 17, wherein the solution is formedfrom magnesium chloride.
 19. The method of claim 1, wherein the one ormore antimicrobial and/or stabilizing agents comprise chlorhexidinegluconate and propylene glycol, a ratio of the chlorhexidine gluconateto the propylene glycol being about 1:50 by weight to about 1:2 byweight.
 20. A method of using a tissue graft, the tissue graft havingbeen submerged in a solution containing one or more antimicrobial and/orstabilizing agents, and stored in the solution for a period of time ofat least 24 hours while the solution is in an unfrozen state, the methodcomprising: implanting the tissue graft in a human or non-human subject.21. The method of claim 1, wherein the tissue graft comprises acellularnerve tissue.